Coordinate expression of anticoagulant heparan sulfate proteoglycans and serine protease inhibitors in the rat ovary: a potent system of proteolysis control.

During the reproductive cycle, ovarian follicles undergo major tissue-remodeling involving vascular changes and proteolysis. Anticoagulant heparan sulfate proteoglycans (aHSPGs) are expressed by granulosa cells during the development of the ovarian follicle. The function of aHSPGs in the ovary is unknown, but they might be involved in proteolysis control through binding and activation of serine protease inhibitors. To identify functional interactions between aHSPGs and heparin-binding protease inhibitors in the follicle, we have coordinately localized aHSPGs, antithrombin III, protease nexin-1, and plasminogen activator inhibitor-1 in the rat ovary during natural and gonadotropin-stimulated cycles. Anticoagulant HSPGs were visualized by autoradiography of cryosections incubated with 125I-antithrombin III, and protease inhibitors were assessed by immunohistochemistry and Northern blot hybridization. Anticoagulant HSPGs were expressed in follicles before ovulation, were transiently decreased in postovulatory follicles, and were abundant in the corpus luteum, mainly on capillaries. Anticoagulant HSPGs were colocalized with protease nexin-1 in follicles from the early antral stage until ovulation, with antithrombin III in the preovulatory stage and after ovulation, and with plasminogen activator inhibitor-1 in the corpus luteum. These data demonstrate that aHSPGs are critically expressed in the ovary to interact sequentially with protease nexin-1, antithrombin III, and plasminogen activator inhibitor-1 during the cycle. The specificity of these inhibitors is shifted toward thrombin inhibition in the presence of heparin, suggesting that aHSPGs direct their action to control fibrin deposition in the follicle. The occupation of aHSPGs antithrombin-binding sites by mutant R393C antithrombin III, injected in the ovarian bursa, decreased ovulation efficiency, further supporting the involvement of aHSPGs in the ovulation process.     

Human follicular fluid heparan sulfate contains abundant 3-O-sulfated chains with anticoagulant activity

Anticoagulant heparan sulfate proteoglycans bind and activate antithrombin by virtue of a specific 3-O-sulfated pentasaccharide. They not only occur in the vascular wall but also in extravascular tissues, such as the ovary, where their functions remain unknown. The rupture of the ovarian follicle at ovulation is one of the most striking examples of tissue remodeling in adult mammals. It involves tightly controlled inflammation, proteolysis, and fibrin deposition. We hypothesized that ovarian heparan sulfates may modulate these processes through interactions with effector proteins. Our previous work has shown that anticoagulant heparan sulfates are synthesized by rodent ovarian granulosa cells, and we now have set out to characterize heparan sulfates from human follicular fluid. Here we report the first anticoagulant heparan sulfate purified from a natural human extravascular source. Heparan sulfate chains were fractionated according to their affinity for antithrombin, and their structure was analyzed by 1H NMR and MS/MS. We find that human follicular fluid is a rich source of anticoagulant heparan sulfate, comprising 50.4% of total heparan sulfate. These antithrombin-binding chains contain more than 6% 3-O-sulfated glucosamine residues, convey an anticoagulant activity of 2.5 IU/ml to human follicular fluid, and have an anti-Factor Xa specific activity of 167 IU/mg. The heparan sulfate chains that do not bind antithrombin surprisingly exhibit an extremely high content in 3-O-sulfated glucosamine residues, which suggest that they may exhibit biological activities through interactions with other proteins.     

Syndecan-4 expression is associated with follicular atresia in mouse ovary

Ovarian granulosa cells synthesize heparan sulfate proteoglycans (HSPGs), that have anticoagulant properties. Moreover, HSPGs greatly increase in the granulosa cells during follicular atresia. However, the species of ovarian HSPGs have not yet been identified. Syndecan-4 (ryudocan, amphiglycan) is a membrane-spanning HSPG and a member of the syndecan family. Herein, we demonstrate that syndecan-4 is expressed in the granulosa cells of type 4-5b follicles and, most intensely, in those of the atretic follicles in the mouse ovary, as revealed by in situ hybridization. There is no relationship between syndecan-4 expression and age or sexual cycle stage. Compared with syndecan-4 expression, syndecan-1 and -3 are expressed more abundantly in postovulatory follicles and the corpora lutea, but less in the type 4-5b follicles and much less in the atretic follicles. Immunohistochemistry also demonstrates syndecan-4 expression in atretic follicles with apoptosis. The present study has revealed the distinct modes of expression of the syndecan family members, and the association of syndecan-4 expression and apoptosis in ovarian atretic follicles.     

Differential partition of anticoagulant heparan sulfate proteoglycans synthesized by endothelial and fibroblastic cell lines

The heparan sulfate proteoglycans that bind and activate antithrombin III (aHSPGs) are synthesized by endothelial cells as well as other nonvascular cells. We determined the amounts of cell surface–associated and soluble aHSPGs generated by the rat fat pad endothelial (RFP) cell line and the fibroblast (LTA) cell line. The RFP cells exhibit higher levels of cell surface–associated aHSPGs as compared to LTA cells, whereas LTA cells release larger amounts of soluble aHSPGs as compared to RFP cells. After confluence RFP cells show an increase in both cell surface–associated and soluble aHSPGs. In contrast, postconfluent LTA cells maintain a constant level of cell surface–associated and soluble aHSPGs. These observations indicate that different cells types can preferentially accumulate aHSPGs as cell surface–associated or soluble forms which could reflect alternate biological functions.     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.     

Cell-specific expression and regulation of soluble guanylyl cyclase alpha 1 and beta 1 subunits in the rat ovary

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and carbon monoxide, resulting in cGMP production. Recent studies indicate that NO and cGMP influence ovarian functions. However, little information is available regarding the ovarian expression of sGC. The present study examined sGC alpha(1) and beta(1) subunit protein levels in the ovary during postnatal development, gonadotropin-induced follicle growth, ovulation, and luteinization as well as in cultured rat granulosa cells. In postnatal rats, sGC alpha(1) subunit immunoreactivity was high in granulosa cells of primordial and primary follicles on Day 5 but low in granulosa cells of larger follicles on Days 10 and 19. Theca cells of developing follicles, but not stromal cells, also demonstrated moderate sGC alpha(1) immunoreactivity. In gonadotropin- treated immature rats, intense sGC alpha(1) subunit staining was similarly observed in granulosa cells of primordial and primary follicles, but such staining was low in granulosa cells of small antral follicles and undetectable in granulosa cells of large antral and preovulatory follicles. Following ovulation, corpora lutea expressed moderate sGC alpha(1) immunoreactivity. Similar ovarian localization and expression patterns were seen for sGC beta(1), indicating regulated coexpression of sGC subunits. Immunoblot analysis revealed no change in total ovarian sGC alpha(1) and beta(1) subunit protein levels during gonadotropin treatment. Similarly, no effect of FSH on sGC subunit protein levels was apparent in cultured granulosa cells. These findings indicate regulated, cell- specific patterns of sGC expression in the ovary and are consistent with roles for cGMP in modulating ovarian functions.     

Regulation of the follicular hierarchy and ovulation

Studies are discussed which investigate the regulation of follicular maturation and the ovulation sequence of the domestic hen. The number of FSH receptors of ovarian granulosa cells decreases as the follicle matures, and this decrease in receptor number is paralleled by a gradual loss of FSH-stimulable adenylyl cyclase (AC) activity. By contrast, LH-stimulable AC activity increases as the follicle progresses through the hierarchy. In addition, FSH stimulates progesterone secretion by granulosa cells of the smaller preovulatory follicles, whereas these cells are only minimally responsive to LH. These data suggest that the maturation of less mature (smaller) follicles is primarily controlled by FSH, while LH may serve primarily as the ovulation-inducing hormone. The ability of LH to stimulate progesterone release and induce premature ovulation is dependent upon the stage of the sequence. Injection of ovine LH 12 hr prior to ovulation of the first (C1) egg of the sequence induces fully potentiated preovulatory plasma progesterone surges and 100% premature ovulation, whereas injection prior to the second (C2) ovulation of the sequence fails to stimulate prolonged progesterone release and induces premature ovulation in less than 50% of injected hens. These results are consistent with data obtained in vitro which suggest that granulosa cells obtained 12 hr prior to a C1 ovulation secrete more progesterone in response to chicken LH compared to those obtained 12 hr prior to the C2 ovulation. These data are discussed in terms of the ovary's ability to act as a regulator of the ovulatory cycle.     

Catecholamine content of the preovulatory follicles of the domestic hen

The role of catecholamines in ovarian function of the domestic hen has not been examined extensively. The aim of this study was first to determine the location of catecholamines in the preovulatory follicle of the domestic hen. Second, norepinephrine (NE), epinephrine (EPI) and dopamine (DA) were measured in the isolated theca layer of the five largest preovulatory follicles at specific times during the ovulatory cycle and changes in catecholamine content were correlated with ovarian events. The five largest preovulatory follicles were removed from chickens at 24, 18, 12, 6 and 2 h before ovulation of the largest (F1) follicle. Theca and granulosa layers were isolated, frozen, weighed and prepared for measurements of catecholamines by the double isotope radio-enzymatic assay. Catecholamines were localized primarily in the theca layer with only small amounts present in the granulosa layer. Norepinephrine was present in the theca layer in concentrations 6- and 30-fold those of EPI and DA, respectively. The content of NE and EPI in the theca layer of the F1 follicle was significantly (p less than 0.01) higher at 6 h before ovulation than at other times for the F1 follicle. In contrast, NE and EPI content of the theca layer of second (F2) and third (F3) largest follicles did not change during the ovulatory cycle. The content of DA was elevated (p less than 0.05) at 12 h before ovulation in F1 and F2 follicles. There was a significant reduction in NE in the theca layer of the fifth largest (F5) follicle between 24 and 18 h before ovulation of the F1 follicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of follicular maturation on 3-hydroxy-methylglutaryl coenzyme A reductase activity in hen granulosa cells

The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase: EC 1.1.1.34) was measured in a microsomal preparation of the granulosa of rapidly growing ovarian follicles of laying hens in the late preovulatory period (2-3 h before expected ovulation). The specific activity of the enzyme was measured in the five largest (F1-F5) preovulatory follicles, F1 being the follicle destined to ovulate first. Enzyme activity increased concomitantly with follicle size. The apparent Km of the enzyme decreased 60-80% from the smallest to the largest preovulatory follicle. There was no significant change in the Vmax during follicle development. Although our results have demonstrated the presence of HMG/CoA reductase in chicken granulosa cells and the progressive increase of its activity with follicular maturation, the quantitative significance of de-novo synthesized cholesterol as steroid hormone precursor remains to be ascertained.     

Patterns of expression of steroidogenic enzymes during the first wave of the ovine estrous cycle as compared to the preovulatory follicle

The expression patterns of steroidogenic enzymes in ovarian antral follicles at various stages of growth in a follicular wave have not been reported for sheep. Ovaries were collected from ewes (n=4-5 per group) when the largest follicle(s) of the first wave of the cycle, as determined by ultrasonography, reached (i) 3 mm, (ii) 4 mm, (iii) > or =5 mm in diameter or when there was a single (iv) preovulatory follicle in the last wave of the cycle, 12h after estrus detection. The expression pattern of steroidogenic enzymes was quantified using immunohistochemistry and grey-scale densitometry. The expression of CYP19 in the granulosa and 3beta-HSD and CYP17 in the theca increased (P<0.01) progressively from 3 to > or =5 mm follicles in the first wave of the cycle and was lower (P<0.01) in the preovulatory follicle compared to > or =5 mm follicles. However, the expression of 3beta-HSD in the granulosa increased (P<0.05) from 3 to > or =5 mm follicles and was maintained (P<0.05) at a high level in the preovulatory follicles. The amount of CYP19 in the granulosa of the growing follicles correlated positively (r=0.5; P<0.03) with the concurrent serum estradiol concentrations. We concluded that the expression pattern of steroidogenic enzymes in theca and granulosa of follicles growing in each wave in the ewe, paralleled with serum estradiol concentrations, with the exception that concentrations of 3beta-HSD in granulosa increased continuously from follicles 3mm in diameter to the preovulatory follicle.     

Expression of prostasin serine protease and protease nexin-1 (PN-1) in rhesus monkey ovary during menstrual cycle and early pregnancy.

Serine proteases and their cognate serpin-class inhibitors are involved in the controlled proteolytic events during follicular development, ovulation, formation, and maintenance of the corpus luteum (CL). In this study, we investigated the expression patterns of prostasin serine protease and protease nexin-1 (PN-1), a serine protease inhibitor also called serpin-E2, in rhesus monkey ovaries during the menstrual cycle and early pregnancy, by using in situ hybridization and immunohistochemistry. Expression of prostasin was localized in oocyte, granulosa cells, and/or theca cells of early antral follicles and antral follicles, with high levels observed in preovulatory follicles. Prostasin was also localized at high levels of abundance in the CL during the menstrual cycle and early pregnancy. During the menstrual cycle, PN-1 was coordinately localized with prostasin in oocytes, granulosa cells, and theca cells of antral follicles and preovulatory follicles and in the CL. In addition, the PN-1 expression level in macaque CL during early pregnancy increased as pregnancy proceeded. We propose that prostasin may be involved in follicular development, ovulation, and CL formation, whereas PN-1 may be present to regulate the proteolysis in these processes.     

Localization of preovulatory expression of plasminogen activator inhibitor type-1 and tissue inhibitor of metalloproteinase type-1 mRNAs in the rat ovary.

Proteinases and their inhibitors control follicular connective tissue remodeling associated with follicular rupture. We examined the regulation and cellular localization of plasminogen activator inhibitor type-1 (PAI-1) and tissue inhibitor of metalloproteinase type-1 (TIMP-1) mRNAs by in situ hybridization. [35S]UTP-labeled RNA probes were hybridized to ovarian sections of eCG-primed immature rats treated with hCG. Before hCG stimulation of ovulation, very low expression of PAI-1 mRNA was observed in theca cells. After hCG administration, expression of PAI-1 mRNA was increased in theca cells of most antral follicles, whereas expression in granulosa cells was limited to preovulatory follicles and only to areas where the basal membrane was dissociated. Before hCG treatment, low expression of TIMP-1 mRNA was observed in theca cells, but not in granulosa cells. After hCG treatment, TIMP-1 mRNA was greatly stimulated in theca cells irrespective of follicle size, while the expression in granulosa cells was limited to large antral follicles. The present study demonstrates cell-specific expression of PAI-1 and TIMP-1 mRNAs in the LH/hCG-stimulated ovary, thus confirming the localized control of preovulatory proteolysis by coexpression of both enzymes and their respective inhibitors.     

A comparison of hormone levels in follicle-lutein-cysts and in normal bovine ovarian follicles

Insulin-like growth factors 1 and 2 (IGF-1 and 2), oxytocin, progesterone, estradiol and ubiquitin were measured in bovine follicle-lutein-cysts and in follicular fluid after the classification of ovarian follicles by size (Class I = <4 mm; Class II = 5-8 mm; Class III = 9-12 mm; Class IV = preovulatory; Class V = cystic). It was found that IGF-1 concentrations increased during growth from 280 ng/ml in small follicles to 489 ng/ml in preovulatory follicles; IGF-2 appeared to remain constant in follicular fluid and in cysts (275 ng/ml). Oxytocin values were low in Class I, II and III follicles (30 pg/ml) but increased in preovulatory and cystic follicles (75 pg/ml). Estradiol increased significantly only in preovulatory follicles. Ubiquitin, a protein reflecting cellular replicative activity, could be found in bovine follicular fluid in high concentrations: 1.6 mug/ml in Class I,II and III follicles with the highest amounts in preovulatory follicles (2.3 mug/ml). In contrast with normal follicles, cysts were found to have a minimal concentration of ubiquitin (0.3 mug/ml). Progesterone levels were 5 times higher in cysts (325 ng/ml) and IGF-1 concentrations were markedly higher in cystic follicles (881 ng/ml) than in the other follicles. Simultaneously, maximum gene expression for IGF-1 was found in granulosa/lutein cells of cystic follicles (Class V), suggesting de novo synthesis of IGF-1. Between the different follicle classes progesterone, oxytocin and IGF-1 concentrations correlated positively (r=0.82). Hormonal levels in follicle-lutein-cysts indicated an arrested stage of insufficient luteinization as a possible result from the premature release of LH or from the release of amounts of LH inadequate to cause ovulation.     

Adenylyl cyclase system of the small preovulatory follicles of the domestic hen: responsiveness to follicle-stimulating hormone and luteinizing hormone

The purpose of this study was to determine if the granulosa cells of the small preovulatory follicles of the domestic hen are a target tissue for follicle-stimulating hormone (FSH). The third largest (F3), fourth largest (F4), and fifth largest (F5) follicles were removed from hens at 20, 12, 6 and 2 h before ovulation of the F1 follicle. Basal, FSH- and luteinizing hormone (LH)-stimulable adenylyl cyclase (AC) activities were measured in the granulosa cells. Isolated granulosa cells of the F5 follicle, obtained 20 h before ovulation of the F1 follicle, were incubated with ovine (o) or turkey (t) FSH and progesterone (P4) was assayed in the medium. Basal AC activity was similar for F5, F4 and F3 granulosa cells except for an increase (P less than 0.01) in F3 follicles removed 2 h before ovulation of the F1 follicle. The FSH-stimulable AC activity of F5, F4 and F3 granulosa cells was elevated over basal (P less than 0.01). The greatest responsiveness was seen in the F5 follicle and the least in the F3 follicle. LH-stimulable AC activity was absent in the F5 follicle but present in the F4 and F3 follicles with the greater responsiveness in the F3 follicle. Isolated F5 granulosa cells secreted significant amounts of P4 in response to oFSH and tFSH. The data indicate that: 1) FSH stimulates the AC system of granulosa cells of the smaller preovulatory follicles (F5 greater than F4 greater than F3) while LH stimulates the AC system of granulosa cells of the larger follicles (F3 greater than F4), and 2) FSH promotes P4 production by granulosa cells of F5 follicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of intracellular degradation of proteoglycans by leupeptin in rat ovarian granulosa cells

Previous work (Yanagishita, M., and Hascall, V. C. (1984) J. Biol. Chem. 259, 10270-10283) has indicated that heparan sulfate (HS) proteoglycans in rat ovarian granulosa cells are degraded by two kinetically distinct pathways. Pathway 1 degrades proteoglycans rapidly with a t 1/2 approximately 25 min without generating appreciable degradative intermediates. Pathway 2 degrades proteoglycans more slowly with a t 1/2 approximately 4 h, generating distinct degradative intermediates: single HS chains of Mr = approximately 10,000 and approximately 5,000. Effects of leupeptin, an inhibitor of thiol proteases, on the intracellular degradation of proteoglycans in the rat ovarian granulosa cell culture were examined using various chase protocols after labeling cells with [35S]sulfate. The presence of leupeptin at 100 micrograms/ml in the culture medium inhibited the intracellular degradation of proteoglycans by approximately 80% during a 7-h chase period after a 20-h labeling. Leupeptin affected neither the cellular content nor the in vitro activities of beta-hexosaminidase and arylsulfatase. Structural analyses of heparan sulfate species in leupeptin-treated cells demonstrated that the drug inhibited the degradation of HS proteoglycans at two distinct points. First, degradation of the core protein was partially inhibited and delayed before the start of glycosaminoglycan degradation. This resulted in the accumulation of degradative intermediates with partially degraded core proteins bearing intact glycosaminoglycan chains. This establishes the initial sequence for HS proteoglycan degradation, with proteolysis preceding endoglycosidase digestion, and suggests that these two degradation steps may occur in physically separate compartments. Second, the final depolymerization of HS fragments through pathway 2 was totally inhibited, resulting in the continuous accumulation of Mr = 5,000 HS chains. This is not due to the direct inhibition of the lysosomal exoglycosidase and sulfatase enzymes responsible for the complete depolymerization of HS chains, since pathway 1, while slowed, continued to completely depolymerize the HS chains in the presence of leupeptin. The results suggest that the intracellular compartment which completely degrades heparan sulfate chains is separate from those containing partially, endoglycosidically processed heparan sulfate chains and that leupeptin interfered with the translocation of glycosaminoglycans to the final degradation site.